The purpose of this MPI proposal between the Goldhaber and Ottolia laboratories is to improve our mechanistic understanding of how sodium-calcium exchange (NCX), the dominant calcium (Ca) efflux mechanism in cardiac cells, functions to regulate cellular Ca, which in turn controls contractility and pacemaker activity. Our labs have been studying the exchanger for more than two decades, despite a number of challenges: 1) there is no proven specific blocker of NCX that can be administered extracellularly; 2) NCX current is sometimes difficult to interpret because the transporter is influenced not only by voltage, but also by temperature, pH and the intracellular and extracellular concentrations of Na and Ca; 3) intracellular Ca is not only transported by the exchanger, but also serves a regulatory function that can influence transport activity. In addition to our longstanding expertise isolating and studying NCX, for this proposal we have generated new highly innovative NCX mouse models: a tamoxifen-inducible cardiac knockout of NCX, which allows us to investigate how Ca regulation adapts over time to the acute removal of NCX in the adult mouse; a unique pH- insensitive NCX knockin mouse to investigate the physiological impact of NCX pH regulation; and the first ventricular-specific plasma membrane Ca pump 1 (PMCA1) KO mouse to determine the relative contribution of NCX and PMCA to Ca homeostasis and EC coupling. Our three specific aims are to study: 1. Atrial-Specific NCX KO?Effects on Nodal Rhythm and Atrial EC Coupling; 2. Acute and Chronic Adaptations of Ventricular EC Coupling and Ca regulation to Genetically Altered Levels of NCX; 3. NCX pH dependence ? implications for EC coupling and arrhythmia. These aims will test the hypotheses that NCX is an essential component of atrioventricular node (AVN) conduction and impulse generation, that acute ablation of NCX in adult mice activates a Ca regulatory and EC coupling adaptation program that is distinct from chronic adaptation, that PMCA1 is a critical alternative to NCX as a Ca efflux mechanism, and that pH sensitivity of NCX is critical for maintaining Ca regulation under conditions of low pH. Our approach is to use our existing and new innovative mouse models, along with state-of-the-art single cell and tissue electrophysiology combined with high speed subcellular Ca imaging techniques, high-quality proteomics and next generation RNA sequencing, to determine how NCX contributes to cardiac function through regulation of Ca. When completed, these studies will improve our mechanistic understanding of the role of NCX and related Ca handling proteins in cellular Ca regulation, EC coupling, and cardiac pacemaker (SAN and AVN) function. Such information is critical to develop effective and safe approaches to improving contractility and cellular pacemaker function in cardiac diseases such as heart failure with reduced ejection fraction, and high degree heart block from AVN disease.